Publications by authors named "Rainer Kaufmann"

34 Publications

Correlative super-resolution fluorescence and electron cryo-microscopy based on cryo-SOFI.

Methods Cell Biol 2021 19;162:253-271. Epub 2020 Dec 19.

Centre for Structural Systems Biology, Hamburg, Germany; Department of Physics, University of Hamburg, Hamburg, Germany. Electronic address:

The combination of super-resolution fluorescence microscopy and electron microscopy at ambient temperatures has become an established technique and a broad variety of modalities are now available to the cell biology community. In contrast, correlative cryogenic super-resolution fluorescence and electron microscopy (super-resolution cryo-CLEM) is just emerging. Aside from technical challenges, one of the major issues is the risk of devitrification of the specimen caused by the laser intensities required for super-resolution imaging. Cryo-SOFI (cryogenic super-resolution optical fluctuation imaging) allows the reconstruction of super-resolution images at particularly low laser intensities. It is fully compatible with the standard sample preparation for cryogenic electron microscopy (cryo-EM) and fairly easy to implement in any standard cryogenic fluorescence microscope.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.021DOI Listing
December 2020

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Cell 2020 01 9;180(2):323-339.e19. Epub 2020 Jan 9.

Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK. Electronic address:

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
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http://dx.doi.org/10.1016/j.cell.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978801PMC
January 2020

Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.

Proc Natl Acad Sci U S A 2019 03 26;116(11):4804-4809. Epub 2019 Feb 26.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, OX3 7BN Oxford, United Kingdom;

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.
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http://dx.doi.org/10.1073/pnas.1810690116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421404PMC
March 2019

The Impact of Sex Differences on Odor Identification and Facial Affect Recognition in Patients with Schizophrenia Spectrum Disorders.

Front Psychiatry 2018 31;9. Epub 2018 Jan 31.

Biopsychosocial Corporation BioPsyC, Vienna, Austria.

Background: Social interactive functions such as facial emotion recognition and smell identification have been shown to differ between women and men. However, little is known about how these differences are mirrored in patients with schizophrenia and how these abilities interact with each other and with other clinical variables in patients vs. healthy controls.

Methods: Standardized instruments were used to assess facial emotion recognition [Facially Expressed Emotion Labelling (FEEL)] and smell identification [University of Pennsylvania Smell Identification Test (UPSIT)] in 51 patients with schizophrenia spectrum disorders and 79 healthy controls; furthermore, working memory functions and clinical variables were assessed.

Results: In both the univariate and the multivariate results, illness showed a significant influence on UPSIT and FEEL. The inclusion of age and working memory in the MANOVA resulted in a differential effect with sex and working memory as remaining significant factors. Duration of illness was correlated with both emotion recognition and smell identification in men only, whereas immediate general psychopathology and negative symptoms were associated with emotion recognition only in women.

Conclusion: Being affected by schizophrenia spectrum disorder impacts one's ability to correctly recognize facial affects and identify odors. Converging evidence suggests a link between the investigated basic and social cognitive abilities in patients with schizophrenia spectrum disorders with a strong contribution of working memory and differential effects of modulators in women vs. men.
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http://dx.doi.org/10.3389/fpsyt.2018.00009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797769PMC
January 2018

A combined 3D-SIM/SMLM approach allows centriole proteins to be localized with a precision of ∼4-5 nm.

Curr Biol 2017 Oct;27(19):R1054-R1055

Micron Oxford Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, Oxford, UK. Electronic address:

Centrioles are small barrel-shaped structures that form centrosomes and cilia [1]. Centrioles assemble around a central cartwheel comprising the Sas-6 and Ana2/STIL proteins. The amino termini of nine Sas-6 dimers form a central hub of ∼12 nm radius from which nine dimer spokes radiate, placing the Sas-6 carboxyl termini at the outer edge of the ∼60 nm radius cartwheel [2]. Several centriole proteins are distributed in a toroid around the cartwheel, and super-resolution light microscopy studies have measured the average radii of these ∼100-200 nm radius toroids with a 'precision' - or standard deviation (s.d. or 1σ) - of ±∼10-40 nm. The organization of Ana2/STIL within the cartwheel, however, has not been resolvable. Here, we develop methods to calculate the average toroidal radius of centriolar proteins in the ∼20-60 nm range with a s.d. of just ±∼4-5 nm, revealing that the amino and carboxyl termini of Ana2 are located in the outer cartwheel region.
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http://dx.doi.org/10.1016/j.cub.2017.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640508PMC
October 2017

Correlative In-Resin Super-Resolution Fluorescence and Electron Microscopy of Cultured Cells.

Methods Mol Biol 2017 ;1663:163-177

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.

Correlative super-resolution light and electron microscopy (super-resolution CLEM) is a powerful and emerging tool in biological research. The practical realization of these two very different microscopy techniques with their individual requirements remains a challenging task. There is a broad range of approaches to choose from, each with their own advantages and limitations. Here, we present a detailed protocol for in-resin super-resolution CLEM of high-pressure frozen and freeze substituted cultured cells. The protocol makes use of a strategy to preserve the fluorescence and photo-switching capabilities of standard fluorescent proteins, such as GFP and YFP, to enable single-molecule localization microscopy (SMLM) in-resin sections followed by transmission electron microscopy (TEM) imaging. This results in a fivefold improvement in resolution in the fluorescence image and a more precise correlation of the distribution of fluorescently labeled molecules with EM ultrastructure compared with conventional CLEM.
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http://dx.doi.org/10.1007/978-1-4939-7265-4_14DOI Listing
May 2018

Preserving the photoswitching ability of standard fluorescent proteins for correlative in-resin super-resolution and electron microscopy.

Methods Cell Biol 2017 5;140:49-67. Epub 2017 May 5.

University of Oxford, Oxford, United Kingdom.

There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed. This chapter details the method we developed for in-resin super-resolution CLEM using single molecule localization microscopy (SMLM) with standard fluorescent proteins (e.g., GFP and mVenus). The key to this approach is being able to preserve not only the fluorescence, but also, and more importantly, the photoswitching ability of the fluorescent proteins throughout the EM sample preparation procedure. Cells are cryofixed using high pressure freezing for optimal structural preservation and then freeze substituted in tannic acid, which preserves the photoswitching ability of the fluorescent proteins and is essential for high-quality SMLM imaging. Resin sections are then imaged using SMLM, achieving a structural resolution of 40-50nm and a localization precision of ∼17nm, followed by transmission electron microscopy. This produces high quality correlative images without the use of specialized fluorescent proteins or antibodies.
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http://dx.doi.org/10.1016/bs.mcb.2017.04.001DOI Listing
February 2018

Structural Basis for Plexin Activation and Regulation.

Neuron 2016 Aug 7;91(3):548-60. Epub 2016 Jul 7.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom. Electronic address:

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.
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http://dx.doi.org/10.1016/j.neuron.2016.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980550PMC
August 2016

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

Sci Rep 2016 06 6;6:27290. Epub 2016 Jun 6.

Micron Oxford Advanced Imaging Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.
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http://dx.doi.org/10.1038/srep27290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893670PMC
June 2016

Towards correlative super-resolution fluorescence and electron cryo-microscopy.

Biol Cell 2016 Sep 22;108(9):245-58. Epub 2016 Jun 22.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.
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http://dx.doi.org/10.1111/boc.201600008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524168PMC
September 2016

SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy.

Sci Rep 2015 Nov 3;5:15915. Epub 2015 Nov 3.

Department of Biochemistry, University of Oxford, Oxford, UK.

Three-dimensional structured illumination microscopy (3D-SIM) is a versatile and accessible method for super-resolution fluorescence imaging, but generating high-quality data is challenging, particularly for non-specialist users. We present SIMcheck, a suite of ImageJ plugins enabling users to identify and avoid common problems with 3D-SIM data, and assess resolution and data quality through objective control parameters. Additionally, SIMcheck provides advanced calibration tools and utilities for common image processing tasks. This open-source software is applicable to all commercial and custom platforms, and will promote routine application of super-resolution SIM imaging in cell biology.
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http://dx.doi.org/10.1038/srep15915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648340PMC
November 2015

Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

PLoS One 2015 4;10(6):e0128555. Epub 2015 Jun 4.

Institute for Theoretical Physics, Heidelberg University, Philosophenweg 19, 69120, Heidelberg, Germany.

It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are investigated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128555PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456097PMC
May 2016

Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.

Sci Rep 2015 Mar 31;5:9583. Epub 2015 Mar 31.

1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK [2] Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.
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http://dx.doi.org/10.1038/srep09583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379466PMC
March 2015

Fluorescence cryo-microscopy: current challenges and prospects.

Curr Opin Chem Biol 2014 Jun 19;20:86-91. Epub 2014 Jun 19.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address:

Studying biological structures with fine details does not only require a microscope with high resolution, but also a sample preparation process that preserves the structures in a near-native state. Live-cell imaging is restricted mostly to the field of light microscopy. For studies requiring much higher resolution, fast freezing techniques (vitrification) are successfully used to immobilize the sample in a near-native state for imaging with electron and X-ray cryo-microscopy. Fluorescence cryo-microscopy combines imaging of vitrified samples with the advantages of fluorescence labeling of biological structures. Technical considerations as well as the behavior of fluorophores at low temperatures have to be taken into account for developing or adapting super-resolution methods under cryo conditions to exploit the full potential of this technique.
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http://dx.doi.org/10.1016/j.cbpa.2014.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094034PMC
June 2014

Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.

Nano Lett 2014 Jul 4;14(7):4171-5. Epub 2014 Jun 4.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics and ‡Department of Biochemistry, University of Oxford , Oxford, United Kingdom.

We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement.
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http://dx.doi.org/10.1021/nl501870pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092024PMC
July 2014

Spatial distribution and structural arrangement of a murine cytomegalovirus glycoprotein detected by SPDM localization microscopy.

Histochem Cell Biol 2014 Jul 7;142(1):61-7. Epub 2014 Feb 7.

Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120, Heidelberg, Germany.

Novel approaches of localization microscopy have opened new insights into the molecular nano-cosmos of cells. We applied a special embodiment called spectral position determination microscopy (SPDM) that has the advantage to run with standard fluorescent dyes or proteins under standard preparation conditions. Pointillist images with a resolution in the order of 10 nm can be obtained by SPDM. Therefore, vector pEYFP-m164, encoding the murine cytomegalovirus glycoprotein gp36.5/m164 fused to enhanced yellow fluorescent protein, was transiently transfected into COS-7 cells. This protein shows exceptional intracellular trafficking dynamics, moving within the endoplasmic reticulum (ER) and outer nuclear membrane. The molecular positions of gp36.5/m164 were visualized and determined by SPDM imaging. From the position point patterns of the protein molecules, their arrangements were quantified by next neighbour distance analyses. Three different structural arrangements were discriminated: (a) a linear distribution along the membrane, (b) a highly structured distribution in the ER, and (c) a homogenous distribution in the cellular cytoplasm. The results indicate that the analysis of next neighbour distances on the nano-scale allows the identification and discrimination of different structural arrangements of molecules within their natural cellular environment.
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http://dx.doi.org/10.1007/s00418-014-1185-2DOI Listing
July 2014

Quantitative analysis of individual hepatocyte growth factor receptor clusters in influenza A virus infected human epithelial cells using localization microscopy.

Biochim Biophys Acta 2014 Apr 27;1838(4):1191-8. Epub 2013 Dec 27.

Kirchhoff Institute for Physics, University of Heidelberg, D-69120 Heidelberg, Germany; Superresolution Light Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany; Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, D-69120 Heidelberg, Germany. Electronic address:

In this report, we applied a special localization microscopy technique (Spectral Precision Distance/Spatial Position Determination Microscopy/SPDM) to quantitatively analyze the effect of influenza A virus (IAV) infection on the spatial distribution of individual HGFR (Hepatocyte Growth Factor Receptor) proteins on the membrane of human epithelial cells at the single molecule resolution level. We applied this SPDM method to Alexa 488 labeled HGFR proteins with two different ligands. The ligands were either HGF (Hepatocyte Growth Factor), or IAV. In addition, the HGFR distribution in a control group of mock-incubated cells without any ligands was investigated. The spatial distribution of 1×10(6) individual HGFR proteins localized in large regions of interest on membranes of 240 cells was quantitatively analyzed and found to be highly non-random. Between 21% and 24% of the HGFR molecules were located in 44,304 small clusters with an average diameter of 54nm. The mean density of HGFR molecule signals per individual cluster was very similar in control cells, in cells with ligand only, and in IAV infected cells, independent of the incubation time. From the density of HGFR molecule signals in the clusters and the diameter of the clusters, the number of HGFR molecule signals per cluster was estimated to be in the range between 4 and 11 (means 5-6). This suggests that the membrane bound HGFR clusters form small molecular complexes with a maximum diameter of few tens of nm, composed of a relatively low number of HGFR molecules. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.
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http://dx.doi.org/10.1016/j.bbamem.2013.12.014DOI Listing
April 2014

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers.

Ultramicroscopy 2014 Aug 19;143:41-51. Epub 2013 Oct 19.

Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address:

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell.
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http://dx.doi.org/10.1016/j.ultramic.2013.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045203PMC
August 2014

Duration of untreated psychosis in a high-income versus a low- and middle-income region.

Aust N Z J Psychiatry 2013 Dec 24;47(12):1176-82. Epub 2013 Sep 24.

1Department of Psychiatry and Psychotherapy, Division of Social Psychiatry, Medical University of Vienna, Vienna, Austria.

Objective: Most data on duration of untreated psychosis (DUP) derives from high-income countries. An inverse relationship between DUP and income and a longer DUP in low- and middle-income (LAMI) countries has been reported. The aim of this study was to compare DUP in a high-income country with that in a LAMI country using the same methodology.

Methods: The sample consisted of in- and outpatients, aged 15-35 years for the Vienna site and 18-35 years for the Pakistani sites, with first-episode psychosis (FEP). DUP was evaluated using psychiatric interviews, medical charts and the Nottingham Onset Schedule. Differentiated reporting of duration of untreated illness (DUI) from prodrome to start of treatment, and DUP from manifest psychotic symptoms to start of treatment was ensured. Primary outcome measures, DUI and DUP, were measured at a 0.025 level of significance.

Results: Thirty-one FEP patients in Vienna (mean age 20.03 years, SD 4.2) and 60 FEP patients from the Pakistani sites (mean age 26.15 years, SD 5.29) participated. The mean age in Vienna was younger due to the different age range inclusion criteria. The severity of psychopathology was more pronounced in the Pakistani sample. Log DUP was significantly different between groups (i.e. longer in the Pakistani sample (p=0.001)). Log DUI showed a trend for longer duration in the Vienna sample; however, this did not reach statistical significance (p=0.036). The severity of positive psychotic symptoms was associated with length of DUI in both regions.

Conclusion: The longer DUP in Pakistan confirms the need to provide affordable treatment for psychosis for young FEP patients in Pakistan and in other LAMI countries. The relatively long period from prodrome to treatment initiation in both regions underlines the need to further establish low-threshold early intervention strategies in order to increase detection rates and reduce factors limiting patients seeking treatment.
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http://dx.doi.org/10.1177/0004867413504472DOI Listing
December 2013

Structurally encoded intraclass differences in EphA clusters drive distinct cell responses.

Nat Struct Mol Biol 2013 Aug 30;20(8):958-64. Epub 2013 Jun 30.

Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.
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http://dx.doi.org/10.1038/nsmb.2617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3941021PMC
August 2013

Localization microscopy (SPDM) reveals clustered formations of P-glycoprotein in a human blood-brain barrier model.

PLoS One 2012 12;7(9):e44776. Epub 2012 Sep 12.

Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany.

P-glycoprotein (Pgp; also known as MDR1, ABCB1) is the most important and best studied efflux transporter at the blood-brain barrier (BBB); however, the organization of Pgp is unknown. The aim of this study was to employ the recently developed super-resolution fluorescence microscopy method spectral precision distance microscopy/spectral position determination microscopy (SPDM) to investigate the spatial distribution of Pgp in the luminal plasma membrane of brain capillary endothelial cells. Potential disturbing effects of cell membrane curvatures on the distribution analysis are addressed with computer simulations. Immortalized human cerebral microvascular endothelial cells (hCMEC/D3) served as a model of human BBB. hCMEC/D3 cells were transduced with a Pgp-green fluorescent protein (GFP) fusion protein incorporated in a lentivirus-derived vector. The expression and localization of the Pgp-GFP fusion protein was visualized by SPDM. The limited resolution of SPDM in the z-direction leads to a projection during the imaging process affecting the appeared spatial distribution of fluorescence molecules in the super-resolution images. Therefore, simulations of molecule distributions on differently curved cell membranes were performed and their projected spatial distribution was investigated. Function of the fusion protein was confirmed by FACS analysis after incubation of cells with the fluorescent probe eFluxx-ID Gold in absence and presence of verapamil. More than 112,000 single Pgp-GFP molecules (corresponding to approximately 5,600 Pgp-GFP molecules per cell) were detected by SPDM with an averaged spatial resolution of approximately 40 nm in hCMEC/D3 cells. We found that Pgp-GFP is distributed in clustered formations in hCMEC/D3 cells while the influence of present random cell membrane curvatures can be excluded based on the simulation results. Individual formations are distributed randomly over the cell membrane.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3440331PMC
March 2013

Superresolution imaging of transcription units on newt lampbrush chromosomes.

Chromosome Res 2012 Dec;20(8):1009-15

Kirchhoff Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany.

We have examined transcription loops on lampbrush chromosomes of the newt Notophthalmus by superresolution microscopy. Because of the favorable, essentially two-dimensional morphology of these loops, an average optical resolution in the x-y plane of about 50 nm was achieved. We analyzed the distribution of the multifunctional RNA-binding protein CELF1 on specific loops. CELF1 distribution is consistent with a model in which individual transcripts are tightly folded and hence closely packed against the loop axis.
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http://dx.doi.org/10.1007/s10577-012-9306-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3761802PMC
December 2012

Role of aripiprazole in treatment-resistant schizophrenia.

Neuropsychiatr Dis Treat 2012 29;8:235-44. Epub 2012 May 29.

Department of Child and Adolescent Psychiatry, Medical University, Vienna, Austria.

About one third of patients with schizophrenia respond unsatisfactorily to antipsychotic treatment and are termed "treatment-resistant". Clozapine is still the gold standard in these cases. However, 40%-70% of patients do not improve sufficiently on clozapine either. In the search for more efficacious strategies for treatment-resistant schizophrenia, drugs with different pharmacological profiles seem to raise new hopes, but are they valid? The aim of this review was to evaluate the evidence for aripiprazole as a potential strategy in monotherapy or combination therapy for patients with treatment-resistant schizophrenia. The evidence for aripiprazole monotherapy and for the combination of aripiprazole with psychotropics other than clozapine is scant, and no recommendation can be made on the basis of the currently available data. More effort has been made in describing combinations of aripiprazole and clozapine. Most of the open-label and case studies as well as case reports have shown positive effects of this combination on overall psychopathology and to some extent on negative symptoms. Several reports describe the possibility of dose reduction for clozapine in combination with aripiprazole, a strategy that might help so-called "treatment-intolerant" patients. The findings of four randomized controlled trials with respect to changes in psychopathology seem less conclusive. The most commonly found beneficial effects are better metabolic outcomes and indicators of the possibility of reducing the clozapine dose. However, other side effects, such as akathisia, are repeatedly reported. Further, none of the studies report longer-term outcomes. In the absence of alternatives, polypharmacy is a common strategy in clinical practice. Combining aripiprazole with clozapine in clozapine-resistant or clozapine-intolerant patients seems to be worthy of further investigation from the pharmacological and clinical points of view.
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http://dx.doi.org/10.2147/NDT.S13830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373202PMC
August 2012

Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy.

PLoS One 2012 2;7(2):e31128. Epub 2012 Feb 2.

Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany.

Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy.TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM), a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of ∼20 nm, yielding a mean structural resolution of ∼50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter <100 nm and 300-600 nm, respectively) were revealed. For Cld5 the two populations were more separated than for Cld3. Discrimination of non-polymeric molecules and molecules within polymeric strands was achieved. For both subtypes of claudins the mean density of detected molecules was similar and estimated to be ∼24 times higher within the strands than outside the strands.The morphometry and single molecule information provided advances the mechanistic analysis of paracellular barriers. Applying this novel method to different TJ-proteins is expected to significantly improve the understanding of TJ on the molecular level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271094PMC
September 2012

Superresolution imaging of biological nanostructures by spectral precision distance microscopy.

Biotechnol J 2011 Sep;6(9):1037-51

Institute of Molecular Biology, Mainz, Germany.

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far-field methods have been developed allowing a substantial increase of resolution ("superresolution microscopy", or "lightoptical nanoscopy"). This opens an avenue to 'nano-image' intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDM(Phymod) . Generally, this SPDM method is based on high-precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual-color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20-nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).
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http://dx.doi.org/10.1002/biot.201100031DOI Listing
September 2011

COMBO-FISH enables high precision localization microscopy as a prerequisite for nanostructure analysis of genome loci.

Int J Mol Sci 2010 Oct 21;11(10):4094-105. Epub 2010 Oct 21.

Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany; E-Mails: (P.M.); (E.S.); (R.K.); (C.C.).

With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
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http://dx.doi.org/10.3390/ijms11104094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996811PMC
October 2010

Localization microscopy reveals expression-dependent parameters of chromatin nanostructure.

Biophys J 2010 Sep;99(5):1358-67

Institute for Theoretical Physics, Heidelberg University, Heidelberg, Germany.

A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.
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http://dx.doi.org/10.1016/j.bpj.2010.05.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931727PMC
September 2010

Imaging label-free intracellular structures by localisation microscopy.

Micron 2011 Jun 28;42(4):348-52. Epub 2010 May 28.

Applied Optics and Information Processing, Kirchhoff-Institute for Physics, University Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany; Institute for Pharmacy and Molecular Biotechnology, University Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany; Institute for Molecular Biophysics, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA.

Localisation microscopy methods allow to realize a light optical resolution far beyond the Abbe-Rayleigh limit of about 200 nm laterally and 600 nm axially. So far, this progress was achieved using labelling with appropriate fluorochromes and fluorescent proteins. Here, we describe for the first time that optical resolution of cellular structures in the λ/10 range (∼50 nm) can be achieved even in label-free cells. This was obtained using Spectral Precision Distance/Position Determination Microscopy (SPDM), a method based on the general principles of localisation microscopy. Besides a substantial resolution improvement of autofluorescent structures, SPDM revealed cellular objects which are not detectable under conventional fluorescence imaging conditions.
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http://dx.doi.org/10.1016/j.micron.2010.03.006DOI Listing
June 2011
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